Contrast medium for mri

ABSTRACT

Disclosed is an MRI contrast agent which enables direct detection and imaging of exfoliated vascular endothelial sites. The MRI contrast agent comprises an imaging unit which contains an unpaired electron-carrying atom and/or molecule and is capable of amplifying or reducing MRI signals, and a detection unit which is bonded to said imaging unit and is capable of selectively recognizing exfoliated vascular endothelial sites and binding thereto. The detection unit is exemplified by one having a chemical structure expressed by the following formula (I), wherein at least one of R 1 -R 11  is, for example, sulfonic acid group, and X is, for example, a phenyl group which may be substituted:

TECHNICAL FIELD

The present invention belongs to the field of in vivo reagent, andparticularly relates to a novel contrast agent for use in MRI.

BACKGROUND ART

In MRI (Magnetic Resonance Imaging), an intravascular contrast agent isused to ensure high-contrast images. Conventional intravascular MRIcontrast agents are dedicated to observation of an overall vascularsilhouette, i.e., the degree of stricture in the vascular lumen toprovide information for the diagnosis and treatment of vasculardiseases, and they cannot be applied for directly detecting lesion siteswhere the vascular endothelia have exfoliated.

The ability to image the exfoliated vascular endothelia directly as theyare would greatly contribute to early disease detection and treatment.However, there are found almost no MRI contrast agents developed forsuch purpose. A method has been proposed for detecting injured orinflamed vascular sites, in which there are used antibodies againstintegrin or fibrin as targeting moiety in the MRI contrast agents (D. A.Sipkins et al., Nature Medicine, 4, 623-626 (1998); S. Flacke et al.,Circulation, 104, 1280-1285 (2001)). However, such antibody-basedcontrast agents are not practical as they are costly because theyrequire complicated steps to prepare and have to be used in a largeamount.

The object of the invention is to provide a novel MRI contrast agentwhich enables direct detection and imaging of exfoliated vascularendothelial sites and which is easy to prepare and inexpensive to use.

DISCLOSURE OF THE INVENTION

Through extensive studies, the present inventors found substances whichare capable of selectively recognizing exfoliated vascular endothelialsites and binding thereto, and achieved the present invention bycombining a structural unit of such substance with a unit of imagingfunction.

Thus, according to the present invention there is provided a contrastagent for use in MRI, comprising an imaging unit which contains anunpaired electron-carrying atom and/or molecule and is capable ofamplifying or reducing MRI signals, and a detection unit which is bondedto said imaging unit and is capable of selectively recognizingexfoliated vascular endothelial sites and binding thereto.

Specifically, the present invention provides an MRI contrast agent whichis novel in that, differently from the conventional MRI contrast agentswhich are dedicated only to imaging the overall vascular silhouette, itis capable of directly detecting exfoliated vascular endothelial sitesas they are, as well as imaging the sites.

A preferred but non-limiting example of the detection unit of the MRIcontrast agent of the present invention is one having a chemicalstructure expressed by the following general formula (I):

In the formula (I), at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰ and R¹¹ may be independently sulfonic acid group, hydroxyl group oramino group, at least one of R¹, R², R³ and R⁴ may be independently analkyl group or alkoxy group having 1 to 3 carbon atoms, the remainingones of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ which do notfall within any of the above-mentioned functional groups are allhydrogen atoms, and X, if present, represents a phenyl group in which atleast one site may be substituted with an alkyl group or alkoxy grouphaving 1 to 3 carbon atoms.

In the chemical formulae as used in the present specification anddrawings, carbon atoms or hydrogen atoms are sometimes omitted inaccordance with the conventional practice.

The MRI contrast agents according to the present invention are ofoutstanding utility in the early detection and early treatment ofvascular diseases because they are capable of selectively detectingexfoliated vascular endothelia. The MRI contrast agents of the presentinvention can be easily prepared through known synthetic reactions andare low-cost because they are effective when used in a relatively smallquantity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction scheme for synthesizing MRI contrast agents thatare embodiments of the present invention.

FIG. 2 shows the results of an experiment conducted to evaluate theimaging capability of an MRI contrast agent of the present invention, byusing an aortic vascular section extirpated from a pig.

FIG. 3 shows the results of an experiment conducted to evaluate theimaging capability of the MRI contrast agent of the present invention,using a living rat.

FIG. 4 shows the results of measurements of the Gd content, from the MRIcontrast agent, remaining in various organs of a rat.

FIG. 5 illustrates the observation sites of a rat with which an in vivoexperiment was carried out to evaluate the imaging capability of the MRIcontrast agent of the present invention.

FIG. 6 shows MRI images of a rat taken using the MRI contrast agent ofthe present invention and a comparative MRI contrast agent.

FIG. 7 shows graphical representations of digitized MRI signalintensities in the MRI imaging tests conducted on the rat using the MRIcontrast agent and the comparative MRI contrast agent.

BEST MODE FOR CARRYING OUT THE INVENTION

In the formula (I) which expresses the chemical structure of a preferredexample of the detection unit of the MRI contrast agent of the presentinvention, at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ andR¹¹ (hereinafter referred to as R¹-R¹¹) may be independently sulfonicacid group (—SO₃H), hydroxyl group (—OH) or amino group (—NH₂). Morespecifically, either all of R¹-R¹¹ are hydrogen atoms (i.e.unsubstituted), or that at least one of R¹-R¹¹ is sulfonic acid group,hydroxyl group, or amino group. In a case where two or more of R¹-R¹¹are sulfonic acid group, hydroxyl group or amino group, such two or morefunctional groups may be the same or different. It is generallypreferable for realizing increased water-solubility of the MRI contrastagent of the present invention that at least one of R⁴-R¹⁰ is sulfonicacid group (particularly in a case where the imaging unit is of lowhydrophilicity).

In addition, in the formula (I), at least one of R¹, R², R³ and R⁴ maybe independently an alkyl group or alkoxy group having 1 to 3 carbonatoms, between which methyl group is particularly preferable. Theremaining ones of R¹-R¹¹ which do not fall within any of theabove-mentioned functional groups are all hydrogen atoms.

Furthermore, in the formula (I), X need not be present, in which casethe benzene ring as shown in the formula (I) is directly bonded to —NH.X, if present, represent a phenyl group in which at least one site maybe substituted with an alkyl or alkoxy group having 1 to 3 carbon atoms(preferably methyl group).

Thus, preferable examples of the chemical formula of the detection unitof the MRI contrast agent of the present invention include, but are notlimited to, the following chemical formulae (II), (III), (IV), (V) and(VI):

There are no particular restrictions with respect to the imaging unit ofthe MRI contrast agent of the present invention, except that it isrequired to contain an unpaired electron-carrying atom and/or moleculeand be capable of amplifying or reducing MRI signals. More specifically,as the imaging unit of the MRI contrast agent, there can be utilized avariety of known substances capable of enabling imaging in MRI due to afunction such as that of shortening the relaxation time of protons (T₁,T₂). For example, there can be used a chelated complex of a paramagneticmetal ion such as Cd³⁺, Dy³⁺ Eu³⁺, Fe³⁺, Mn²⁺; a nitroxide radicalmolecule such as a piperidine derivative and a pyrrolidine derivative;or a ferromagnetic material such as magnetite (Fe₃O₄). Particularlypreferred is a chelated complex of gadolinium [Cd(III)] in which is usedsuch compound as DTPA, DOTA or EDTA as the ligand.

Thus, as a preferable MRI contrast agent according to the presentinvention, there is exemplified, without limitation thereto, oneexpressed by the following formula (VII) in which the imaging unit isDTPA (diethylenetriaminepentaacetic acid) complex of Gd(III) and thedetecting unit has the composition of the aforementioned chemicalformula (II).

The MRI contrast agent of the present invention can be easilysynthesized by appropriately modifying known reactions. For example, ina case where there is used as the imaging unit a metal complex such asgadolinium chelated complex, the desired MRI contrast agent can beobtained by introducing a Boc group into an amino compound (i.e. byt-butoxycarbonylating the amino compound) which corresponds to a portionof the detection unit expressed by the formula (I), allowing theresultant to react with the ligand portion of the metal complex,removing the Boc group, combining the resultant with the remainingportion of the detection unit through the diazo coupling, and subjectingthe resultant to a complex formation with the metal (see the workingexample set out below).

The MRI contrast agent of the present invention thus obtained is capableof selectively recognizing exfoliated vascular endothelial sites (lesionsites) and binding thereto through the detection unit while enablingsaid sites to be visualized as MRI signals through the imaging unit. TheMRI contrast agent of the present invention can therefore provide veryreliable information in the treatment and diagnosis of vasculardiseases, as a contrast agent specific to exfoliated vascularendothelial sites. For example, it is known that exfoliated vascularendothelial sites are unstable and arteriosclerosis will proceed morerapidly therein than in the sites without such exfoliation even if thedegrees of the strictures are of the same level. The detection ofexfoliated vascular endothelial sites using the MRI contrast agent ofthe present invention therefore contributes to early diagnosis andtreatment of such circulatory diseases.

While working examples are set out below in order to more clearly definethe features of the present invention, the present invention is not inany respect limited to such working examples.

EXAMPLES Example 1 Synthesis of the Contrast Agent

In accordance with the reaction scheme as shown in FIG. 1, there wassynthesized the MRI contrast agent of the present invention as expressedby the aforementioned formula (VII).

(1) Synthesis of Boc DMB (Step A).

Dimethylbenzidine (DMB) 11.0 g (4.71 mmol), dibutoxycarbonylketone(Boc₂O) 1.28 g (5.88 mmol) and triethylamine 0.714 g were added to 20 mlof dichloromethane, followed by stirring at room temperature for 48hours. The reaction mixture was subjected to filtration, added withchloroform, and then washed with a saturated aqueous solution ofL-sodium tartrate to remove unreacted DMB. The chloroform phase wassubjected to concentration in vacuo, refining by silica gelchromatography, and then vacuum drying to obtain the desired product(0.60 g).

(2) Synthesis of DMB-DTPA (Step B).

Boc DMB 0.30 g (0.96 mmol) was dissolved in 20 ml of pyridine. In theresultant solution was suspended DTPA anhydride 0.343 g (0.96 mmol). Theatmosphere of the container (round-bottom flask) was replaced withnitrogen gas and the flask was sealed, followed by stirring the contentsthereof overnight at 50° C. The unreacted DTPA anhydride was removed bysuction filtration. The filtrate was subjected to vacuum drying, addedwith 6 ml of 0.1 NaOH (0.6 mmol), allowed to stand for one hour, andthen lyophilized. The resultant was dissolved in water, and the desiredintermediate was fractionated by ODS/silica gel column chromatography.The fractionate was dissolved in 4 ml of TFA, and the solution wasallowed to stand for thirty minutes and then added with ether 40 ml. TheBoc group was removed by recovering the precipitate to obtain thedesired product at a yield of 0.85 g.

(3) Diazo Coupling (Step C):

A diazonium salt was prepared by admixing DMB-DTPA 65 mg (62.3 μmol) and35% HCl 16.5 μl (187 μmol) in 1 ml of water, and NaNO₂ 4.5 mg (65.2mmol) was added to the mixture, followed by stirring for thirty minutes.Diazo coupling reaction was carried out by adding the salt dropwise into1 ml of aqueous solution containing 21.2 mg (62.2 μmol) of monosodium1-amino-8-naphthol-2,4-disulfonate and 26.4 mg (249 μmol) of Na₂CO₃, onan ice bath. Then, 35% HCl was added dropwise to the reaction mixture toobtain the desired product as the precipitate, at a yield of 42.5 mg.

(4) Complex Formation with Gadolinium:

A sample 19.8 mg of the product of the diazo coupling was dissolved in2.2 ml of water. An equimolar amount of 1M GdCl₃ aqueous solution wasadded to the solution. The pH of the resultant solution was adjusted to7.0 by adding 1M aqueous NaOH solution to obtain an aqueous solution (10mM) containing the desired product (VI).

Example 2 Evaluation of Imaging Capability by MRI (1)

For the contrast agent as synthesized (prepared) in Example 1,evaluation of in vitro imaging capability by MRI was carried out with anaortic vascular section extirpated from a pig, as a specimen.

The aortic vascular section extirpated from a pig was spread in arectangular shape measuring about 2 cm×3 cm to prepare a vascularsection sample. The endothelial surface of the left half of the samplewas exfoliated with a scalpel. The sample was then immersed in a 10 mMaqueous solution of the contrast agent for ten seconds. Followingthorough washing with physiological saline solution, the sample wassubjected to the evaluation by MRI as well as by visual observation.

The results are shown in FIG. 2. FIG. 2 (A) is a picture taken with adigital camera, which corresponds to the visual observation. It wasvisually observed that the exfoliated endothelial site (the left half)was colored blue, suggesting accumulation of the contrast agent on thesite. FIG. 2 (B) is an MRI image (by the T1 weighted spin echo method).FIG. 2 (C) graphically shows MRI signal intensities at the exfoliatedendothelial site and the normal endothelial site, in which the MRIsignal intensities are digitized with a computer-aided image analysissystem (NIHImage). The signal intensity at the exfoliated endothelialsite is about ten times as high as that at the normal endothelial site,demonstrating that the contrast agent of the present invention iscapable of specifically binding to the exfoliated endothelial sitethereby producing a clear image through the MRI signal.

Example 3 Evaluation of Imaging Capability by MRI (2)

The contrast agent of the present invention was evaluated for imagingcapability by means of an ex vivo experiment using a living rat.

A balloon-tip catheter was passed through the left femoral artery of therat to injure the carotid artery with the balloon. Then 2 ml of aqueoussaline solution containing 24 mM of the contrast agent (EBDTPA-Gdprepared in Example 1) was injected to the rat through the right jugularvein. After a predetermined period of time (10, 30 or 120 minutes), theright carotid artery and the left carotid artery were extirpated,developed, washed with a physiological saline, and then subjected toevaluation by MRI. The MRI evaluation was carried out by imaging theextirpated carotid artery sections, under the condition of being addedwith one or two droplets of physiological saline solution, by the T1weighted spin echo method.

The results are shown in FIG. 3. In FIG. 3, by “injured” is denoted theexfoliated endothelial site (the left carotid artery), which had beeninjured by the balloon, while “intact” denotes the normal endothelialsite (the right carotid artery). The photographs of the developedvascular samples as presented in FIG. 3 reveal purple coloring withintensities corresponding to the MRI signal intensities described later.The pictures (A) as presented in FIG. 3 below the photographs of thedeveloped vascular samples illustrate the states of the samples preparedfor the study by MRI. As illustrated, each carotid artery section wasdeveloped on a glass plate and covered with one or two droplets ofphysiological saline solution, for the MRI study. (The leftmost pictureof (A) shows a control composed of only physiological saline solution.)Below (A) in FIG. 3, there are shown MRI images (B) of the respectivesamples and digitized MRI signal intensities (C) (digitized with thecomputer-aided image analysis system, NIHImage, as in Example 2).

As seen from FIG. 3, the signal intensity of the injured site (theexfoliated endothelial site) is 1.74 times as high as that of the normalsite after a lapse of ten minutes from the injection, demonstrating thatthe contrast agent of the present invention is capable of selectivelydetecting and imaging vascular exfoliated endothelial sites even in aliving body. It is also seen that after a lapse of thirty minutes thesignal intensities of the exfoliated endothelial site and the normalsites weakened and the difference in the signal intensity therebetweenshortened and further that after 120 minutes there was observed nosubstantial difference in the signal intensity between the exfoliatedsite and the normal site, indicating that the contrast agent of thepresent invention can be rapidly excreted out of the body.

This was also verified by measuring Gd (gadolinium) content remaining invarious organs of the rat, in which the measurement was carried out asfollows: The rat was administered with 1 ml heparin and a blood samplewas taken from the rat. Then physiological saline solution wascirculated throughout the rat, and the various organs were extirpatedfrom the rat. To each organ and the blood sample was added 3 ml nitricacid, followed by standing for one hour. The resultant was added with25% hydrogen peroxide aqueous solution and then allowed to standovernight at room temperature. The resultant solution was warmed andadded with 6 ml water. The insolubles were removed by filtration, andthe filtrate was analyzed by ICP.

The results of the measurement are shown in FIG. 4. It can be seen fromFIG. 4 that there was substantially no remaining Gd in organs other thanthe blood and kidney, suggesting that the contrast agent of the presentinvention can be mostly excreted as urine through the kidney. In fact,this was ascertained from the fact that, ten minutes after theadministration of the contrast agent into the rat, the urine was coloredpurple due to the contrast agent.

Example 4 Evaluation of Imaging Capability by MRI (3)

This example is to evaluate the imaging capability of the contrast agentof the present invention by means of an in vivo experiment using aliving rat.

A rat (weighing about 300 g) was anesthetized with pentobarbital, andinserted with a balloon-tip catheter through the left femoral artery[FIG. 5 (a), 1]. The left common carotid artery was scraped by theballoon to injure the endothelia thereof. Then the rat was injected,through the left femoral vein [FIG. 5 (a), 2], with 2 ml (about 160μM/kg) of physiological aqueous solution of the contrast agent asprepared in Example 1 (24 mM). At predetermined lapses of time, imageswere taken by MRI (the T1 weighted spin echo method) around the rightand left common carotid artery [the cross section along the line N-N inFIG. 5 (a)]. The rat was supplementally anesthetized in order to keep itmotionless during the imaging operation. For comparison, an evaluationexperiment was also carried out in the same manner with a conventionalagent, DTPA-Gd.

The results of the imaging operation are shown in FIG. 6 and FIG. 7.FIG. 6 shows the images of the rat taken in the vicinity of the commoncarotid artery at the cross section corresponding FIG. 5 (b). In FIG. 6,by “injured” is designated the left common carotid artery injured by theballoon, corresponding to 4 in FIG. 5 (b), while by “intact” isdesignated the right common carotid artery, which is normal andcorresponds to 5 in FIG. 5 (b). The numeral 3 in FIG. 5 (b) denotes thebronchium. There was observed a clear MRI signal in the “injured” leftcommon carotid artery when the contrast agent of the present inventionwas used (as can be seen from the clear white area indicated by anarrow), whereas no such signal was observed when the conventionalcontrast agent, DTPA-Gd, was used.

This fact is further demonstrated in FIG. 7 showing the time-varying MRIsignal intensities of the injured common carotid artery and the normalcommon carotid artery, in which the signal intensities are digitized (inthe same manner as in Example 2 and Example 3). Specifically, in thecase where the contrast agent of the present invention was used, the MRIsignal intensity for the injured left common carotid artery was higherthan that for the normal right common carotid artery at ten minutesafter the administration of the agent, and it increased with time,leveling off in about one hour after the administration. By contrast,with the conventional MRI agent, DTPA-Gd, no substantial difference inthe signal intensity was observed between the injured common carotidartery and the normal common carotid artery, making it impossible todetect any injured sites.

INDUSTRIAL UTILIZABILITY

The MRI contrast agent of the present invention is a new type ofcontrast agent which is capable of directly detecting and imagingexfoliated vascular endothelial sites, thereby contributing to the earlydetection and early diagnosis of vascular diseases.

1. A contrast agent for use in MRI, comprising an imaging unit whichcontains an unpaired electron-carrying atom and/or molecule and iscapable of amplifying or reducing MRI signals, and a detection unitwhich is bonded to said imaging unit and is capable of selectivelyrecognizing exfoliated vascular endothelial sites and binding thereto.2. An MRI contrast agent as claimed in claim 1, in which the detectionunit is one having a chemical structure expressed by the followinggeneral formula (I):

wherein at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹may be independently sulfonic acid group, hydroxyl group or amino group,at least one of R¹, R², R³ and R⁴ may be independently an alkyl group oralkoxy group having 1 to 3 carbon atoms, the remaining ones of R¹, R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R¹¹ which do not fall within any ofthe above-mentioned functional groups are all hydrogen atom, and X, ifpresent, represents a phenyl group in which at least one site may besubstituted with an alkyl group or alkoxy group having 1 to 3 carbonatoms.
 3. An MRI contrast agent as claimed in claim 2, in which thedetection unit is one having a chemical structure expressed by thefollowing chemical formula (II), (III), (IV), (V) or (VI).


4. An MRI contrast agent as claimed in any of claim 1, in which theimaging unit comprises a chelated complex of gadolinium.
 5. An MRIcontrast agent as claimed in claim 4, which is expressed by thefollowing chemical formula (VII):


6. An MRI contrast agent as claimed in claim 2, in which the imagingunit comprises a chelated complex of gadolinium.
 7. An MRI contrastagent as claimed in claim 6, which is expressed by the followingchemical formula (VIII):


8. An MRI contrast agent as claimed in claim 3, in which the imagingunit comprises a chelated complex of gadolinium.
 9. An MRI contrastagent as claimed in claim 8, which is expressed by the followingchemical formula (IX):